Allozymes

The enzyme extraction was performed on muscle and liver tissue of each fish according to the protocols described by Pasteur et al. (1987). The desired enzyme loci were highlighted by colour-adapted protocols by Berrebi et al. (1993, 1995) and Tsigenopoulos et al. (1999). Ten enzyme systems (IDH, LDH, PGI, MDH, 6PGD, AAT, GDA, AK and PGM) and the soluble muscle proteins (PT), corresponding to 25 presumptive loci, were tested. Table 1a presents details of the retained loci and buffers used (nomenclature according to Shaklee et al. 1990).

Introns

DNA was extracted according to the classical phenol/ chloroform protocol (Sambrook et al. 1989). EPICPCR used primers complementary to exon sequences flanking introns. Exon sequences are mostly conserved among the species, providing universal priming zones which hold a practical interest (Atarhouch et al. 2003). A total of 27 primer pairs were tested in this study. Table 1 lists the characteristics of the retained loci for the analysis. PCR reactions were performed using an Eppendorf Mastercycler in a volume of 10 µL for each individual, containing 1 µL of 10X buffer (Promega), 2.5 mM MgCl 2, 0.2 mM of each dNTP (Invitrogen), 0.5 µM of each of the two primers, one of which is labeled with a fluorochrome (Cy3, Cy5 or Fuorescein, MWG-Biotech), 0.3 U of Taq polymerase (Sigma) and 1 µL of DNA template at about 150 µg/mL.

Table 1.

Marker descriptions.

a) Allozymic loci: tissue of extraction, electrophoretic buffers and staining solution compositions.

The PCR program included an initial denaturation at 94 °C for 3 min, then 35 cycles consisting of denaturation for 1 min, binding of the primers (annealing) at the temperature specific to each locus (see Table 1b) for 1 min and extension at 72 °C for 1 min 20 sec; the program ended with a final extension at 72 °C for 10 min.

The PCR products were then subjected to electrophoresis on acrylamide denaturing (urea) 8 % gel. This gel was read by a Hitachi FMBIO II scanner. The FMBIO Analysis 8.0 software can accurately measure allele sizes in number of base pairs (bp).

Statistical analyses

The data matrix was statistically processed involving three steps:

1) Calculation of the parameters commonly used in population genetics (allele frequencies, heterozygosity).

2) Calculation of Wright's fixation indices such as Fis (checking panmixia) by the estimator f of Weir & Cockerham (1984) and linkage disequilibrium (Black & Krafsur 1985). The statistical significance of these parameters was estimated by resampling permutations tests (5000 permutations). The Genetix software (Belkhir et al. 1998) was used for these first two steps. 3) The clonality of genotypes was tested using the Geneclone software (Arnaud-Haond & Belkhir 2007). This software established the probability of existence for each multilocus genotype taking into account the Fis of the population analyzed, i.e. a deviation from panmixia as described by Young et al. (2002). Next, the probability that n repetitions of the same genotype can occur in the population by n independent events of sexual reproduction was estimated (psex; Tibayrenc et al. 1990, Parks & Werth 1993, Arnaud-Haond et al. 2005). If psex < 0.05, repeated genotypes have a significant probability of being propagated by unisexual reproduction.

Table 2.

Allelic frequencies and heterozygosities of the five loci analyzed. Ho = observed heterozygosity, He = expected heterozygosity.

Polyploidy compensates low polymorphism

Polyploidy is considered to confer more tolerance to ecological variations because the duplication of their genes provides metabolic flexibility similar to a permanent high genomic heterozygosity (Uyeno & Smith 1972, Otto & Whitton 2000, Jiang et al. 2013). The Labeobarbus fritschi Moroccan populations showed low polymorphism and thus low heterozygosity; however their hexaploidy may compensate this lack. Technically speaking, hexaploidy provoked a very low level of usable markers in our study due to complex multiple bands patterns at all nuclear markers.

El Gharbi (1994) analyzed a population of L. fritschi from the River Zerouka in her work on the phylogeny of the genus Barbus s.l. complex in North Africa. Her sample came from a scientific station located downstream of the Allal El Fassi dam lake analyzed in this study. It showed that the downstream population was in HWE. This author does not seem to have estimated the sex ratio of this river population.

In this 1994 study, only two allozyme loci were polymorphic (AK-2* and PGI-4*) out of the 15 analyzed in total. In this study, five loci were evidenced as polymorphic among 8 allozymic and 15 intronic interpretable markers. The polymorphic loci analyzed in the two studies were similarly scarce (respectively 2/15 and 5/23).

Sex migration dimorphism hypothesis

In agreement with the observations of Bouhbouh (2002), examination of the gonads of 39 barbel fish of 2002 from the dam lake revealed that they were all females. How can this population reproduce with only females in the lake? The two relevant hypotheses that may explain this phenomenon are: migration of male fish with seasonal meetings with females, or unisexual reproduction.

The absence of males as remarked by Bouhbouh (2002) was observed after many gillnet fishing expeditions and during all seasons between 2000 and 2001. Our own observations on a sample of 2002 are consistent with this result. If the meeting of the two sexes was seasonal, multiple fishing opportunities should have revealed some males. It is therefore very likely that no male is living in the lake. This simple observation leads us to consider unisexual reproduction as the best hypothesis.

Unisexual reproduction hypothesis

The data analysis resulted in some important features pointing in favour of the unisexual reproduction hypothesis. Deviations from HWE are important population features. Firstly, the Fis estimate showed that there was a very significant excess of heterozygotes while most panmictic deviations described in natural populations showed a deficit in heterozygotes, often due to a Walhund effect (Aurelle et al. 2002). Secondly, we found two pairs of loci (PGI-2*/Mlc2a and PGI- 4*/RP) that were statistically associated (p < 0.001). Analysis of genotypes showed that two thirds of the specimens possessed the same genotype at the five loci studied. Given the low polymorphism at our disposal, it is questionable whether such sets of identical genotypes can be observed in fish populations that reproduce sexually. The test of clonality (Geneclone) showed that at least one multilocus genotype, repeated four times (four individuals, n°4, 14, 25 and 36, bearing the letter h in the Appendix), had a significant probability of not being propagated by sexual reproduction processes. All these results lead us to the unisexual reproduction hypothesis.

Gynogenesis

Unisexual female populations are known in fish, often after episodes of hybridization between species of the same genus (see review in Chevassus 1998). The cases of the genera Poecilia and Poeciliopsis illustrate two classical patterns of female monosex populations. Poecilia formosa lives in unisexual female populations, reproducing by gynogenesis (premeiotic endomitosis) with the production of diploid eggs. The sperm of another species is only used to trigger the embryonic development without participating in the genome of the future embryo as evidenced in several studies (Hubbs & Hubbs 1932, Turner et al. 1980, Castelli 1994, Ryan et al. 1996, Yang et al. 2001).

Reproduction by gynogenesis at the most maintains the initial heterozygosity (Schultz 1977, Bulger & Schultz 1979) but its reduction is expected. This mode of unisexual reproduction does not meet the Mendelian laws and causes major deviations from HWE and statistical linkages among loci.

In the hybridogenesis case of the Poeciliopsis complex (Moore & McKay 1971, Chevassus 1998), unisexual female populations of P. monacha coexist with bisexual sympatric populations (P. lucida and P. occidentalis). Thus, two forms of unisexual diploid female populations result from the hybridization between females of P. monacha and males of each of the two sympatric bisexual species. In these diploid females, meiosis is not recombinant but reductional and hemiclonal (hybridogenesis), allowing the genome of the mother to be transmitted without recombination: the eggs contain only the maternal genome (P. monacha), the paternal genome brought by the males of P. lucida or P. occidentalis participating in the somatic line only (Schultz 1980). Hemiclonal hybridogenesis maintains their hybrid status (Schultz 1977, Bulger & Schultz 1979), leading to highly polymorphic populations.

In this Labeobarbus monosex population, the trigging role of sympatric and even syntopic males of Luciobarbus setivimensis can be expected since favorable spawning areas in the dam lake are probably limited, leading to accidental gametes contacts and since Luciobarbus genus is a distant lineage (Labeobarbus is an African hexaploid lineage while Luciobarbus is a North African and European tetraploid one). According to the limited information produced here for the Labeobarbus case, gynogenesis is more probable than hybridogenesis which should follow fecondation between very different lineages (tetraploid and hexaploid of a distinct origin, Tsigenopoulos et al. 2002).

Limited nuclear genetic data is mainly due to the hexaploid nature of the Labeobarbus genus (Tsigenopoulos et al. 2002). Demonstrating the mechanism of unisexual reproduction occurring in the lake requires much more information than that produced for this preliminary study. Microsatellites and introns are traditionally the main markers used in this kind of research. Despite the high number of intron system runs (15), only two proved to be interpretable for this study. It is probable that microsatellites would add a more few markers, but genome scan methods will clearly be necessary (genome scan techniques as AFLP) to disentangle the process.